Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.


RXFP2 expression in adrenal gland
Adrenal gland from hypertensive and normotensive Caucasian men (n = 6 each) were studied.Samples from individuals with evidence of hypertension, heart disease, or cardiovascular disease were excluded from the normotensive cohort.Individuals with cancer were excluded from both the hypertensive and normotensive cohorts (Supplementary Table S1).Relative gene expression analysis conducted via RT-qPCR revealed that adrenal RXFP2 mRNA is more abundant by 3.16-fold in hypertensive compared to normotensive men (p = 0.041) (Fig. 1e).Regional bias of tissue collection was ruled out by using genetic markers of adrenal cortex (NOV) and adrenal medulla (CHGB) 39 .No significant difference in the gene expression of NOV or CHGB between adrenal tissue samples from normotensive and hypertensive samples was detected (Supplementary Fig. S5).

Small molecule RXFP2 antagonist discovery
A high throughput screen (HTS) assay was developed using HEK293 cells transduced with RXFP2 via baculovirus-mediated gene transfer (BacMam) 40 .RXFP2 antagonists were identified using a primary, single-shot antagonist screen (10 µM) of the GSK compound library.Putative 'hits' were confirmed by 11-point dose-response curve with 1:3 dilutions from 100 µM.The primary assay was performed using homogenous time resolved fluorescence (HTRF) to detect compound inhibition of INSL3 EC 80 -induced cAMP in HEK293 expressing human RXFP2 (hRXFP2; INSL3 EC 50 = 0.048 nM).The HTS campaign identified 104 hits across 8 clusters showing blockade of hRXFP2.
The HTS output was biased for lipophilic, poorly soluble compounds.The majority of hits initially identified had a property forecast index (PFI) value greater than 6, which correlates to poor drug developability outcomes 41 .This may indicate the preference of RXFP2 to bind large, peptide-like molecules over small molecule chemicals.Removing compounds with high PFI and low efficacy (PFI > 6, < 40% inhibition of 10 nM INSL3 cAMP activity) was implemented to reduce lipophilicity.Resulting molecules were counter screened against parental HEK293 to remove compounds inhibiting cAMP with < tenfold selectivity compared to RXFP2 expressing cells.An orthogonal readout was utilized to confirm RXFP2 antagonist activity; whereby RXFP2 was transiently expressed in HEK293 stably expressing a member of the G αq G protein family (murine G α16 ) to induce promiscuous G-protein coupling and subsequent calcium mobilization.This FLIPR assay detected compounds that inhibit INSL3-induced intracellular calcium (INSL3 EC 50 = 13.44 nM).

DNA-encoded library technology
To increase the diversity of RXFP2 antagonists, affinity-based DNA-Encoded Library (DEL) techniques were applied to identify compounds that bind to the entire RXFP2 ECD.The DEL campaign identified 37,563 potential binders.Prioritized compounds were resynthesized off-DNA (Supplementary Figs.S8-S11), but ECD binders were inactive in the HEK-hRXFP2 and HEK-rRXFP2 cAMP assays halting further progression (Table 1, Fig. 2).Recent models of RXFP2 predict that INSL3 binding to the ECD induces a receptor confirmation whereby the LDLa module directly interacts with extracellular regions of RXPF2 transmembrane loops to initiate receptor signaling 18,21 .The present study suggests that small molecule binding of the ECD alone is not sufficient to antagonize INSL3-induced cAMP generation.

RXFP2 monoclonal antibody discovery
Monoclonal antibodies were explored as an alternate modality to neutralize RXFP2 activity.Although RXFP2 has a large ECD for antibody-based targeting, the ECD is highly conserved between mouse and rat, which might hinder a mouse immunization strategy.The sequence identity between rat and mouse for the LDLa-linker, LRR, and ECL domains are 97%, 95%, and 100%, respectively.The sequence identity between rat and human for the LDLa-linker, LRR, and ECL domains are 81%, 83%, and 87%, respectively.The Alphafold prediction models of rat LDLa-linker and LRR domains suggest that the majority of non-conserved residues are on the convex face of the LRR domain which is the non-INSL3 binding side (Supplementary Fig. S12).To account for all potential epitopes of RXFP2, a DNA immunization strategy was employed using constructs encoding the full-length rat and human RXFP2 protein in conjunction with tolerance breaking peptides 42 and immunomodulators 43 .Using a hybridoma workflow, a panel of 47 anti-rRXFP2 antibodies was generated.Thirty-eight of the discovered antibodies bind to the LDLa-linker of the ECD (Fig. 3a).Sequencing and clonotyping revealed that the anti-RXFP2 antibodies consisted of 26 unique clonotypes.Picking one representative clone from each clonotype, 26 antibodies were recombinantly expressed in a rat IgG2b backbone to evaluate their affinities and neutralizing activities.The binding kinetics of the antibodies against rat and human LDLa-linker proteins were determined (Fig. 3b,c).www.nature.com/scientificreports/ The antibodies had a wide range of affinities against the rat LDLa-linker protein (Table 2).Only antibodies, 4B1, 4F6, and 4G6, had cross reactivity to the human LDLa-linker (Fig. 3c).RXFP activity assays were developed in HEK293 with stable expression of RXFP2 or RXFP1.RXFP2 antagonists were identified using a primary, three-point antagonist screen at INSL3 EC 80 -induced cAMP generation in HEK293-rRXFP2 cells (INSL3 EC 50 = 8.4 nM).Putative hits were confirmed by 11-point dose-response curve up to 1 µM mAb.Additional profiling was conducted in INSL3 EC 80 -induced cAMP generation in HEK293-hRXFP2 (EC 50 = 2.9 nM) and relaxin EC 80 -induced cAMP generation in HEK293-rat RXPF1 (EC 50 = 29.5 nM).See Supplementary Fig. S14for INSL3 and relaxin-induced cAMP dose responses.Seventeen antibodies were blockers of INSL3-stimulated rRXFP2 signaling and most had potencies in the double-digit nM range (Table 2, Representatives in Fig. 3d and e, Supplementary Table S2).Only two antibodies, 4G6 and 4B1, showed strong blockade of hRXFP2 (Fig. 3e).These RXFP2 mAbs were selective with no blockade of rRXFP1 except for 4G7 (Fig. 3f) and did not cause rRXFP2 agonism (Fig. 3g).
To better understand the diversity of epitopes that were covered by the RXFP2 mAbs, a bead-based highthroughput epitope binning experiment 44 revealed 4 epitope bins across the 17 antibodies (Supplementary Fig. S13).The human/rat RXFP2 cross reactive antibodies belong to two epitope bins and were further evaluated for binding to the linker-less human LDLa peptide (UnitProt Q8WXD0 43-80) by ELISA.4F6 and 4G6 bind to the LDLa linker-less domain while 4B1 requires the linker domain for binding (Supplementary Fig. S13).The different domain requirements for binding are consistent with results from the binning experiment showing 4F6 and 4G6 are in a separate bin from 4B1.

INSL3 in adrenal cell assays
Activity assays were developed using NCI-H295R, an immortalized human cell line derived from the adrenal cortex that is sensitive to cAMP stimulation and produces corticosteroids 44 .Endogenous RXFP2 mRNA determined by qPCR was too low for a robust assay; therefore, H295R with stable expression of human or rat RXFP2 were generated.The RXFP2 expression level in H295R over-expressing RXFP2 was within the range of the adrenal RXFP2 expression level in rHTN men.The potency of INSL3 on cAMP generation and the expression level of the rate-limiting enzymes for cortisol (11β-hydroxylase, CYP11B1) and aldosterone (aldosterone synthase, CYP11B2) synthesis were determined.INSL3 dose-dependently increased cAMP generation (EC 50 = 4 nM) after 30 min of treatment.After 48 h of treatment, the EC 50 of human INSL3 on CYP11B1 mRNA expression was 1.3 nM (H295R-hRXFP2, Fig. 4b) and 0.3 nM (H295R-rRXFP2).The EC 50 of human INSL3 on CYP11B2 mRNA expression was 2.0 nM (H295R-hRXFP2, Fig. 4c) and 0.4 nM (H295R-rRXFP2).Cortisol and aldosterone from H295R supernatants were measured by ELISA.As shown in Fig. 4d and e, rat INSL3 EC 50 was 0.2 nM for cortisol secretion and 2.2 nM for aldosterone secretion.

RXFP2 mAb rat pharmacokinetics
The pharmacokinetics (PK) of six RXFP2 mAbs (1G1, 2D4, 4B1, 4G6, 1F2, 3F3, 1 mg/kg, sc.) was evaluated in Wistar Han rats over 28 days.As shown in Fig. 6, the rank order of preferred PK profile was 1G1 > 2D4 > 1F2 > 4B1, 4G6, 3F3.The half-life of 4B1 was 6.25 days, and the extrapolated values for 1G1, 2D4, 4G6, 1F2, and 3F3 were ≥ 11 days.Because of their long half-lives, the clearance and volume of distribution could not be accurately determined except for 4B1, which had an apparent clearance of 0.62 ± 0.03 mL/h/kg and apparent volume of distribution of 0.133 ± 0.005 L/kg matching a typical IgG pharmacokinetic profile 46 .

Discussion
Hypertension is a major cause of cardiovascular and renal diseases which are among the highest contributors to morbidity and mortality worldwide, and men up to 65 years old have higher prevalence of hypertension than women.Therefore, the development of new medicines for rHTN is urgently needed to reduce the health and economic burden.Complex traits such as hypertension tend to be highly polygenic and many genetic variants with small effects contribute to the phenotype.Most of the SNPs identified in GWAS for blood pressure are pleiotropic and non-coding, which makes functional mapping challenging.Thus far, approximately 30 genes with rare variants involved in blood pressure dysregulation and > 1477 common SNPs including in the RXFP2 locus have been associated with blood pressure 6,47 .In this study, we identify a novel male-specific genetic association in rHTN and provide evidence for a potential mechanism whereby the target protein, RXFP2, may regulate blood pressure.We report the first potent, selective RXFP2 antagonists for use in rodent studies.
Here, we show genetic evidence for RXFP2 as a potential mechanism for rHTN through a genome-wide association study.Sex-stratified analysis in both 23andMe and UK Biobank show the association only in men indicating that RXFP2 may play a role in the higher blood pressure in men.The shared genetic signal between the rHTN GWAS and the RXFP2 eQTL in the adrenal gland suggest a RXFP2 antagonist would be beneficial for the treatment of rHTN.Experimentally, we validated the variant to gene analysis and show that RXFP2 gene expression is increased in adrenal gland of men with hypertension.Phenome-wide significant associations of the GWAS lead SNP with hyperaldosteronism and other hypertension-related diseases further strengthen the hypothesis for a role of RXFP2 in rHTN.
Human adrenal cell assays were established to assess the functional consequences of RXFP2 agonism and antagonism.Because endogenous RXFP2 gene expression is low in H295R, they were engineered to stably express RXFP2 to increase the signal window across assays.The results show that INSL3, the natural ligand of RXFP2, increases cAMP generation in human adrenal cells, which has been previously associated with increased steroid secretion 48 .Because INSL3 also increases CRE-dependent gene transcription 23 , steroidogenesis was evaluated in H295R stably expressing RXFP2.INSL3 dose-dependently increased 11β-hydroxylase and aldosterone    www.nature.com/scientificreports/synthase transcripts which are the most proximal, rate-limiting enzymes in the synthesis of cortisol and aldosterone, respectively.The impact on steroidogenesis is consistent with previously demonstrated INSL3-stimulated CYP17A1 gene expression in theca cells, which was blocked by siRNA targeted at RXFP2 15 .Further functional evaluation of RXFP2 agonism was necessary to translate the increase in gene transcription to a clinically meaningful effect.Indeed, elevations in circulating concentrations of aldosterone and cortisol are required for the hormones to achieve their pro-hypertensive effects via activation of the mineralocorticoid receptor in target tissues like kidney and brain.Thus, ELISAs were established to measure aldosterone and cortisol in supernatants from H295R stably expressing RXFP2, and INSL3 dose-dependently increased aldosterone and cortisol release.
The increase in steroid hormone secretion is consistent with dibutyryl-cAMP-stimulated aldosterone and cortisol secretion in H295R 48 and INSL3-stimulated androgen secretion in Leydig 14 and theca cells 15 .Since RXFP2 increased adrenal corticosteroids, it may not be surprising to find phenome-wide significant associations of rs2146377 in hyperaldosteronism and rs1535532 in primary aldosteronism 17 , which are associated with hypernatremia, hypokalemia, and hypertension.rs1535532 was one of the top eQTLs for RXFP2 in the adrenal gland and the PheWAS with primary aldosteronism was specific to men 17 .Whether adrenal RXFP2 also causes hypertension through PLC/PKC-medicated increases in aldosterone 49,50 or through catecholamine production remains to be elucidated.
A limitation of GWAS is the possibility that associations are false-positives due to population stratification; however, the risk was mitigated by correction for primary axes of genetic variation and replication from an independent cohort.Observational studies in biobanks have the potential for healthy volunteer bias and rHTN a. were discretely dosed to male Wistar Han rats (1 mg/kg, s.c., n = 3/mAb) once and the plasma concentration of RXFP2 mAbs was determined over 28 days.(b) Rat pharmacokinetic parameters calculated by noncompartmental analysis (NCA) analysis.Values are reported in mean ± standard deviation.ND: not determined due to insufficient data points or AUC extrapolated% > 20.C max is the maximum concentration, T max is the time to the maximum concentration, T 1/2 is the terminal half-life, Cl/F is the apparent clearance, AUC last is the area under the concentration-time profile from time zero to the time of last quantifiable concentration.
may reflect a lack of compliance to medications, leading to possible attenuation in effect size.These limitations underscore the importance of validation and follow-up with functional assays, as was done in this study.
Discovering small molecule antagonists of class A GPCRs such as RXFP2 can be challenging, although there is an example of a RXFP2 agonist 28 .Therefore, we conducted a HTS and a monoclonal antibody campaign to discover potentially both short-acting and long-acting RXFP2 antagonists.The primary assay utilized cAMP generation as a measure of receptor activity in HEK293 cells transiently or stably expressing hRXFP2, an approach similar to what was used in the discovery campaign for RXFP2 agonists 28 .We further enhanced the ability to identify small molecule blockers by application of a DNA encoded library technology.Despite identifying modestly potent and selective RXFP2 small molecule blockers that reduce aldosterone synthase transcript in human adrenal cells, physiochemical properties including ligand efficiency and high lipophilicity limited their drug development opportunity.
The antibody discovery campaign discovered more potent and selective antagonists of both rat and human RXFP2.Some of the RXFP2 mAbs have potencies in the double digit nanomolar range that provide ≥ 85% inhibition of the INSL3-induced cAMP generation in HEK-hRXFP2 and HEK-rRXFP2.Most of them also completely inhibit INSL3-induced gene expression of 11β hydroxylase and aldosterone synthase and representative mAbs also reduce cortisol and aldosterone secretion with similar potency and efficacy as their steroidogenesis effects.The potencies of the RXFP2 mAbs in the adrenal cell assays are greater than in the HEK cAMP assay which may be related to the efficiency of the adrenal cell response, which is supported by the higher INSL3 potency in adrenal cells than in HEK.Across all assays, the RXFP2 mAbs are more potent and efficacious than the small molecule RXFP2 antagonists, which may reflect differences in binding domains.
The RXFP2 blocking antibodies bind to the LDLa-linker domain of RXFP2.Previous studies have highlighted the importance of both the linker and LDLa domains in RXFP2 receptor activation and signaling 9,18,[20][21][22][23] .Removal of the LDLa results in abolished signaling 9,18,23 .Similarly, residues within the linker, GDxxGWxxxF (G46-F55), are required for receptor activation 20,21 .The current data further reinforces the critical role the LDLa-linker domain plays in receptor activation and provides insight into the possible mechanism of action of these novel RXFP2 mAbs.
We propose the RXFP2 mAbs function by interfering with regions critical for receptor activation; however, they use different mechanisms to achieve their antagonistic activity.The human LDLa peptide ELISA and epitope binning facilitated stratification of the 3 cross-reactive human/rat RXFP2 blocking antibodies into two distinct epitope bins (Supplementary Fig. S13).Antibody 4B1 requires the linker for binding; whereas, 4F6 and 4G6 require the LDLa domain for binding.To interrogate the epitopes, AlphaFold, which can accurately predict some antibody:antigen complexes, was employed 51 .High confidence predictions for 4B1 and 4G6 in complex with the rat LDLa-linker domain were obtained, but not for 4F6 (see "Methods" section for details).The structural model of the 4B1 rat LDLa-linker domain complex agrees with the experimental ELISA data, as it shows 4B1 binding to linker at the conserved GDxxGWxxxF motif (Supplementary Fig. S15).This motif has been shown to be crucial for receptor activation, likely by allowing rearrangement of the linker to allow the LDLa domain to interact with the extracellular loops of the transmembrane domain 20,21 .4B1 likely blocks receptor activation indirectly by preventing linker rearrangement.The AlphaFold model for the 4G6 rat LDLa-linker complex is also in agreement with the experimental data and predicts that the 4G6 epitope is mostly located on the LDLa domain (Supplementary Fig. S15).The predicted epitope covers the N-terminus of the first beta-sheet (K8-F11) which has been shown to be crucial for receptor activity 23 .The structural model suggests that 4G6, unlike 4B1, may directly block the interaction between the LDLa domain and the transmembrane domain, which is required for receptor signaling.LDLa domains of RXFP1 and RXFP2 modulate receptor activation via similar mechanisms 52 .However, 4G6 did not block relaxin-induced RXFP1-mediated cAMP generation and chimeric RXFP1/2-mediated activity was not evaluated.
The levels of RXFP2 mAbs that are required for blocking adrenal hormone release are likely achievable in rodents based on pharmacokinetic (PK) studies in rat.Using the mAb concentration at trough, we assumed dose-linearity to estimate the dose and length of time to achieve tenfold higher IC 50 HEK-rRXFP2 cAMP activity, which is higher than what would be required to achieve the IC 50 in the H295R-rRXFP2 steroid hormone assays.2D4 at approximately 9 mg/kg, 1G1 or 1F2 at approximately 10 mg/kg, 4B1 at approximately 1324 mg/kg, and 4G6 at approximately 30 mg/kg could achieve that inhibition for approximately 28 days in rat.
Given the expression of RXFP2 in organs that are important in the regulation of blood pressure, and the previously described functions of INSL3/RXFP2 in the brain 16 , renal glomerular cells 11 , and androgen secretion 14 , we cannot rule out non-adrenal mechanisms for RXFP2 in the regulation of blood pressure.During development, androgens increase RXFP2 expression in gubernaculum which mediates testis descent 53 .Whether androgens increase RXPF2 in adult tissues and is a mechanism for the sex-specific difference in hypertension remains unknown.Additionally, RXFP2 signaling does not initiate β-arrestin-mediated internalization 54 so the lack of desensitization may contribute to rHTN.With the discovery of potent, selective RXFP2 antagonists with good PK, the broader contributions of RXFP2 could be further explored.
Our goal for conducting GWAS in resistant hypertensive individuals was to identify new drug targets and ultimately new medicines for the treatment of rHTN because of its critical importance in the development and progression of CVD and CKD.Direct inhibition of aldosterone synthase lowers blood pressure in rHTN 55 .Whether greater blood pressure reduction could be achieved by blocking RXFP2 which targets multiple mechanisms remains to be tested.Based on historical data and statistical modeling, drug targets with human genetic evidence are 2.6 times more likely to be successful during clinical trials 56 .The current studies provide new genetic evidence in rHTN in men and a role for adrenal RXFP2 activity in rHTN.Whether RXFP2 activity is causal or secondary to rHTN remains to be determined.With the discovery of these RXFP2 antagonists, we have tools to further interrogate the role of RXFP2 in health and disease and to provide promise for developing genetically informed drug treatments for precision medicine in rHTN.).We excluded all third degree relatives (KING > 0.8).UK Biobank data was accessed using application 20,361 and all of the participants provided informed consent.Genetic analysis.Genotyping, quality control, and imputation were performed centrally by UK Biobank.Among the UK Biobank study population, we filtered the genotype data to only include variants with MAF > 1%, INFO score > 0.8, and Hardy-Weinberg equilibrium exact test p value greater than 1e-6.We required a maximum per-variant and per-sample missing call rate < 0.1.The final target dataset included 9.4 million SNPs after excluding all duplicate SNPs.Primary analyses were run using logistic regression in the hail software package comparing cases of treatment-rHTN to controls with non-treatment-rHTN.Secondary analyses were run, also using logistic regression in the hail software package, comparing cases of treatment-rHTN to controls with no hypertension.Both primary and secondary analyses were stratified by sex as defined by UK Biobank.

FinnGen
Phenotyping and genetic analysis methods for the FinnGen cohort have been previously published and all data used in this manuscript are publicly available at https:// r9.finng en.fi.All PheWAS results for rs2146377 are available at https:// r9.finng en.fi/ varia nt/ 13: 31631 505-A-G.We want to acknowledge the participants and the investigators of the FinnGen study.

GTEx eQTL data
The data used for the eQTL analyses described in this manuscript were obtained from the GTEx Portal on October 21, 2023 and/or dbGaP accession number phs000424.v8.p2.

Gene expression analysis in adrenal gland
Frozen adrenal gland tissues from six hypertensive and six normotensive men were acquired under the relevant guidelines and regulations from BioIVT and used in accordance with the corresponding institutional review board approved informed consent form.Tissues were dissociated using GentleMACS M Tubes, followed by total RNA isolation (Qiagen), and cDNA synthesis (Applied Biosystems).qPCR was completed using TaqMan probes for RXFP2 (Hs00373128_m1), CHGB (Hs01084631_m1), NOV (Hs00159631_m1), and ACTB (Hs99999903_ m1).Taqman probes for the adrenal medulla (CHGB) and cortex (NOV) were used to rule out regional bias of samples.For the human adrenal tissue RXFP2 transcript analysis, power analysis was determined from a pilot study for sample size determination.To minimize potential sources of bias, samples for qPCR assay were randomized and blinded and results were tested for normality (GraphPad Prism).Based on the distribution of results, the Mann-Whitney U test was applied and p < 0.05 indicated statistical significance.

HEK-RXFP screening and profiling assays
cAMP generation was measured using the Lance Ultra cAMP kit as per manufacturer's instructions (Perkin Elmer) in HEK293 cells transduced in tissue culture flasks with RXFP BacMam, incubated at 37 °C/5% CO 2 for 18 h before assay.Primary single shot HTS and specificity assays were performed in 1536-well format.HEK293 wild type (wt) or RXFP2-transduced HEK293 cells were detached, washed in PBS and resuspended in assay buffer (HBSS, 5 mM Hepes, 0.1% BSA, 0.5 mM IBMX, pH 7.4).The cell solution was plated at a density of 500 cells/well in 3 µL into white, 1536-well plates pre-stamped with 50 nL of test compound and incubated for 30 min.Agonist stimulation was induced with the addition of a further 2 µL of assay buffer supplemented with a final EC 80 concentration of ligand (20 nM Isoprenaline-no RXFP receptor or 4.5 pM INSL3-RXFP receptor).After a 30 min incubation, 4 µL of Lance detection reagent was added and the FRET signal read 1 h later using a TR-FRET-capable plate reader, with excitation at 340 nm and emission at 615 nm and 665 nm.Results were expressed as acceptor fluorescence (signal at 665 nm/signal at 615 nm) normalized to 100% inhibition and 0% inhibition controls (+/− agonist stimulation respectively).Further confirmation, selectivity and ortholog cAMP assays were performed post-HTS in 384-well format using a modified protocol.Here, 10,000 cells/well were plated in 5 µL into white, 384-well plates pre-stamped with 100 nL of test compound and incubated for 30 min.
Agonist stimulation was induced with the addition of a further 5 µL of assay buffer supplemented with a final EC 80 concentration of ligand (120 nM INSL3-RXFP2, 0.89 nM INSL3-rRXFP2, 1.6 nM H2 Relaxin-RXFP1).After a 30 min incubation, 10 µL of Lance detection reagent was added and results collected as above.
For the RXFP2 orthologue assay, calcium detection was measured using FLIPR assay technology in HEK293-MSRII G α16 cells.Cells were resuspended in fresh cell culture media in the presence of RXFP2 BacMam and seeded at a density of 15,000 cells/well in 50 µL into black clear-bottomed 384-well plates and incubated at 37 °C/5% CO 2 for 24 h prior to assay.Media from wells was aspirated and replaced with 30 µL/well of assay buffer (HBSS, 25 mM HEPES, pH 7.4) supplemented with dye-loading reagents (30 µM probenecid, 3 µM Fluo-4 and 750 µM Brilliant Black).Plates were incubated for 1 h at 37 °C/5% CO 2 .Test compounds were added to dye-loaded cells at 10 µL/well and incubated for 10 min.Agonist stimulation was induced with the addition of 10 µL/well assay buffer supplemented with a final EC 80 concentration of ligand (2.4 nM INSL3).Fluorescence was detected immediately using a FLIPR TETRA plate reader, with excitation at 488 nm and emission at 530 nm.Results were expressed as fluorescence (signal at 530 nm-background read) normalized to 100% inhibition and 0% inhibition controls (−/+ agonist stimulation, respectively).Human INSL3 (Phoenix Pharmaceuticals Inc, 035-27) was used in screening and profiling assays.Dose curve fitting and IC 50 determinations were performed in Activitybase XE (version 9.2, IDBS) using 4-parameter logistic curve (Model 203).

DNA encoded library selection
DEL pools containing 93 libraries were used to screen against the following RXFP2 constructs: human LDLa linker [huRXFP2-LDLa-linker(38-105)-ratFc-8XHIS] and rat RFXP2 LDLa linker [ratRXFP2-LDLa-linker(20-88)-ratFc-8XHIS].Selections were performed in triplicate.In the first rounds of selection, DEL molecules were enriched against their respective target.In the final round of selection, the enriched DEL molecules were split equally across 3 selection conditions: (1) human LDLa-linker_rat Fc or rat LDL-linker_rat Fc, (2) rat Fc (control for LDLa_linker), and (3) buffer only.In a round of selection, the DEL pool was incubated with 1 mM of protein at 25 °C for 45 min in 100 µL of selection buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 2.5 mM CaCl 2 , 0.10% Triton X-100, 10 mM Imidazole, 0.1 mg/mL ssDNA).Following incubation, the protein was captured on 50 µL slurry of IMAC magnetic beads (equal volumes of Invitrogen Dynabeads His Tag slurry and Pierce Ni-NTA magnetic bead slurry) pre-washed with selection buffer.While bound to the magnetic beads, the samples were washed twice with selection buffer.Following the washes, beads were re-suspended in 60 µL of elution buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 2.5 mM CaCl 2 , 0.10% Triton X-100, 10 mM Imidazole).In all but the last round, a heat elution step (95 °C for 10 min) was performed to recover the target bound molecules before starting the next selection round with fresh protein.Recovered DEL samples were analyzed using SYBR Green qPCR on a Roche LightCycler in order to determine how many PCR amplification cycles were required for DNA sequencing.Following the last round of selection, heat elution was not performed.Samples on IMAC resin were resuspended in molecular biology grade water and amplified by PCR.PCR reactions were cleaned up with AMPure resin using 1.8× equivalents of resin to PCR product.Sequencing was performed on an Illumina Novaseq.
Selections with the extracellular domain construct (TEV-Flag-6XHis-HiBit-homo sapiens RXFP2) were performed with DEL pools containing 98 libraries pooled with equal copies of every small molecule.Triplicate selections were performed as described above in selection buffer containing 50 mM potassium phosphate pH 7.4, 300 mM NaCl, 1 mM CHAPS, 10 mM imidazole, 0.1 mg/mL ssDNA.Selections were repeated in the same buffer with the addition of 5% inositol.Elution was performed in buffer containing 50 mM potassium phosphate, pH 7.4, 300 mM NaCl, 1 mM CHAPS, and 10 mM imidazole.After selection, enriched DEL molecules were PCR amplified, AMPure treated, quantified, and sequenced as described above.

Hybridoma development
Spleens and lymph nodes were harvested from DNA immunized mice three days following the final boost.Final boosts for mice was either 25 µg each hu and rat LDLa-linker-rFc or irradiated rRXFP2 HEK293 stable cells (Rad Source RS-2000).Splenocytes and lymphocytes from immunized mice were fused with SP2/mIL-6 mouse myeloma cells (American Type Culture Collection CRL-2016) by electrofusion (Nepagene ECFG21) and incubated at 37°C in the presence of 5% CO 2 overnight in ClonaCell-HY Medium C (Stemcell 03803).The following day the cells were harvested and prepared for plating into ClonaCell-HY Medium D (Stemcell 03804).Each 45 million fused lymphocytes was incubated with 1.0 mg of 0.2 µm syringe filtered sodium azide free antimouse IgG Fc fragment specific secondary-FITC reagent (Jackson Immunoresearch Cat#115-096-071), 10 mL total volume.The mixture was incubated for 5 min in the dark at room temperature.The 10 mL volume was then combined with 90 mL of ClonaCell-HY Medium D. The mixture was then shaken well and incubated for 5 min incubation, in the dark, at 37 °C.Finally, 100 mL of the mixture was plated into 6 omnitray plates (Nunc 140156) and hybridoma colonies were allowed to grow for ~ 7 days post fusion after which the omnitray plates were imaged on the Clonepix 2 colony picker (Molecular Devices) and the IgG + hybridomas were picked into 96-well plates containing 200 µL/well of ClonaCell-HY Medium E (Stemcell 03805).The IgG + hybridomas were allowed to grow in 96-well plates for ~ 7 days, upon which the supernatants were screened for RXFP2 specificity on a flow cytometer (CytoFlex LX).

Flow cytometry analysis of hybridoma clones
Sera from Balb/c and Swiss-Webster mice was taken four days following the fourth, sixth, eighth, eleventh, and thirteenth injection of RXFP2 DNA and screened on HEK293 parental, HEK293-rRXFP2, HEK293-hRXFP2.Hybridoma supernatants and purified antibodies were screened on HEK293 parental, HEK293-rRXFP2, and HEK293-hRXFP2.Cells were dissociated from flasks using non-enzymatic Corning CellStripper (25-056-C), resuspended in media, counted, centrifuged at 1200 rpm for 5 min, washed with 1× PBS, and added to 96-well U-bottom plates (Corning 3365) at 500,000-1,000,000 cells per well.Sera were diluted 1:100 and then serially diluted 1:2 in PBS containing 5% FBS (R&D Systems S12450H); hybridoma supernatants were screened neat.Samples were added to cells (50 µL/well) and incubated on ice for 60 min.Plates were centrifuged (1200 rpm, 5 min, 4 °C) and washed three times with PBS/5% FBS (200 µL/well).APC-Goat mouse IgG Fc fragment specific (Jackson Immunoresearch 115-136-071; 1:100 diluted in PBS) was then added and the plates incubated on ice in the dark for 60 min.Plates were again centrifuged and washed three times with PBS/5% FBS (200 µL/well), and a final resuspension of 150 µL/well of PBS was performed.The plates were then read using a Cytoflex LX flow cytometry (Beckman Coulter).Median fluorescence intensity (MFI) of each sample was then measured using the FlowJo software (Treestar, Inc.).

ELISA for antibody screening
Rat LDLa-linker_ratFc, human LDLa-linker_ratFc, or ratFc, or human LDLa peptide (Q8WXD0 43-80) was diluted to 1 µg/mL in 1X PBS and immobilized in separate 96-well ELISA plates (Nunc Maxisorp), 50 µL/well.The plates were then incubated overnight at 4 °C.The following day the plates were washed three times with ELISA wash buffer (PBS + 0.05% Tween-20) and then blocked with ELISA blocking buffer (PBS + 0.05% Tween-20 + 1% BSA), 200 µL/well, and incubated 1 h at room temperature on a shaker.Plates were again washed three times followed by the addition of the primary antibodies.Hybridoma supernatant was added neat, 50 µL/well; mouse serum was added 1:100 and then serially diluted 1:2 for 8 points, 50 µL/well.Purified antibodies were added 5 µg/mL in the first well and serially diluted down the plate 1:2 for 8 points, 50 µL/well.Primary antibody was incubated for 1 h at room temperature on a shaker.Plates were again washed three times followed by the addition of the secondary antibody; HRP-Goat anti mouse Fab2 fragment specific antibody (Jackson Immunoresearch, 115-036-006) or HRP-Goat anti rat Fab2 fragment specific antibody (Jackson Immunoresearch 112-036-072) was added to the plates 1:5000, 50 µL/well, incubated 1 h at room temperature on a shaker.Plates were washed a final three times and Turbo-TMB ELISA (Thermo 34022) reagent was added, 50 µL/well, incubated 10 min at room temperature on a shaker.Stopping solution (2N Sulfuric acid) was added to the plates 50 µL/ well, and then read on a plate reader (SpectraMax Molecular Devices) at 450 nm.

In vitro antibody epitope binning
Biotinylated Rat LDLa-linker_ratFc protein was captured at a concentration of 2 µg/mL to lumavidin beads (Luminex).Capture was performed for 1 h at room temperature in the dark.Beads were then washed 2× with flow buffer (1× PBS + 2% FBS) by centrifugation 3 min at 3500 rpm; wash buffer was aspirated and beads were diluted to a concentration of 2.5 × 10 6 beads/mL.Protein coated beads were then incubated with each antibody in the panel (herein referred to as benchmark) at 5 µg/mL of antibody with 1.25 × 10 6 beads/mL for 45 min at room temperature in the dark (one benchmark mAb per lumavidin bead type).Beads were again washed 2 × with flow buffer by centrifugation.After wash all bead types were pooled and made to 1.25 × 10 6 beads/mL.All test panel antibodies were then prepared in a 96-well v-bottom plate, 20 µL/well of antibody at 3.3 µg/mL concentration.The pooled beads were then added to the v-bottom plate, 40 µL of pooled beads per well and then incubated with the test panel antibodies for 45 min at room temperature in the dark.Beads were again washed 2 × with flow buffer by centrifugation.Detection antibody (Jackson Immunoresearch, G anti Ms-FITC) was then added at 5 µg/mL, 50 µL/well for 15 min at room temperature in the dark.Beads were again washed 2× with flow buffer by centrifugation.Final resuspension of beads was performed with 50 µL of flow buffer and then plates were read.

AlphaFold multimer prediction
AlphaFold multimer v2.2.4 (doi.org/https://doi.org/ 10. 1101/ 2021.10. 04.463034) was used to generate a prediction of antibody:rat LDLa-linker (Uniprot entry: Q5ECL0 residues 19-88) complex for 4B1, 4F6, and 4G6.Twenty-five predictions were generated for each antibody:antigen complex, and model quality was evaluated based on their respective iPTM scores.Previous work 50 has shown accurate antibody:antigen complex predictions are possible and an interface predicted modeling score (iPTM) score threshold of 0.75 corresponds to a possible high confidence model cutoff.While we obtained an iPTM score of 0.76 and 0.82 for the 4B1:antigen and (Lipofectamine LTX) with plasmids encoding rat and human RXFP1 and RXFP2.Supernatants containing the viral particles were collected and filtered through a 0.45 µm filter (Corning 431220) before concentrated with the Lenti-X Concentrator (Takara 631231).Sub-confluent NCI-H295R cells were then transduced with each of the viral supernatants before limiting dilution was used to seed single cell clones into 96-well plates.Each clone was visually inspected and expanded into 24-well plates before cryopreservation and qPCR screening.The same approach was used to generate stable HEK293 lines expressing human and rat RXFP1 and RXFP2 cDNA.The cAMP assay was conducted using the Eurofin Hithunter kit per manufacturer's instructions in a 384-well format.All dose curve fitting and EC 50 and IC 50 determinations were performed in Prism (v9.3.0) using the non-linear fit, variable slope (four parameter) function.

Adrenal cell steroidogenesis assays
Cells were prepared as described above and used for assays.55,000 cells were detached and seeded into each well of a 96-well plate.Replica plates at 3.33× final concentration of the test compound were made and cells were treated with test compounds or mAbs for 1 h before human INSL3 at approximately EC 80 (270 pM) was added to the wells.The final test compound or mAb concentrations ranged from 10 mM to 15 nM (in 1% DMSO or PBS).Cells and the conditioned supernatant were collected at 48 h post treatment.
Total RNA was prepared using the MagMax MirVana Total RNA isolation kit (Applied Biosystems, A27828), and cDNA synthesis was performed per manufacturer's protocol.Using 25 ng cDNA per reaction, qPCR was performed in technical duplicates.Probes for CYP11B1 (Hs01596404_m1), CYP11B2 (Hs01597732_m1), and GAPDH (Hs02786624_g1) were analyzed.RT-qPCR data was analyzed using the ΔΔCt method.ΔCt was calculated by subtracting internal GAPDH control Ct from the gene Ct.ΔΔCt was calculated by subtracting the average ΔCt of all negative control samples.Gene expression fold change (FC) was calculated as FC = 2 (−ΔΔCt) .Dose curve fitting was performed in Prism (v9.3.0) using the variable slope (four parameter) function.Data entered as XY table, with x = [Inhibitor] (nM) and y = CYP11B2 or CYP11B1 fold change.The data was fitted with top concentration excluded, as we observed a reduction at the top vehicle (DMSO) dose (1%).The data was fitted by constraining the bottom equal to the bottom of the corresponding INSL3 dose curve fit for the set.Individual dose point data was normalized to the corresponding DMSO dose point for the corresponding DMSO control.

Adrenal cell corticosteroid hormone secretion assays
The day after seeding, H295R cells were treated and incubated for 48 h before supernatants and cell lysates were collected.Treatments included INSL3, IgG, 1F2, and 4G6 at 0.035 pM-3.5 µM, 2D4 at 0.01 nM-1 uM, or phosphate buffered saline control.In RXFP2 antagonist assays, 3.5 nM INSL3 was used as the stimulus.Cortisol present in the adrenal cell supernatant at 48 h post treatment was detected by ELISA (R&D KGE008B).Sample dilution was optimized at 1/90 using a standard curve.Aldosterone was below the limit of detection by ELISA (R&D KGE016) and required liquid-liquid extraction (LLE) twice using methyl tetra butyl ether.Post extraction samples were dried (SPE Nitrogen dryer) and reconstituted for ELISA analysis.All samples were run in duplicates.Dose curve fitting and EC 50 and IC 50 determination were performed in Prism (v9.3.0 or v9.5.1) using the non-linear fit, variable slope (four parameter) function.

Animals
Rats and mice used in this study were maintained in a facility adhering to AAALAC international guidelines.All experiments were approved and performed in compliance with the Institutional Animal Care and Use Committee at 23andMe or GSK and complied with the ARRIVE guidelines.
Mice (Balb/c and Swiss-webster, Jackson Laboratory) were 2-3 months old when they were used in the RXFP2 antibody campaign.For generation of anti-RXFP2 antibodies, mice were administered a priming dose of 10 µg mFlt3L, followed by weekly dosing with 2.5 µg mGM-CSF, 25 mg human full-length RXFP2 DNA, and 25 mg rat full-length RXFP2 DNA (HTV) and boosted (6 doses of hRXFP2 DNA, 15 doses of rRXFP2 DNA) based on titers from test bleeds.All doses were prepared and administered in the following manner: total DNA was prepared in lactated Ringer's solution volume equivalent to 10% mouse body weight and delivered via hydrodynamic tail vein injections administered over 4-8 s.Serum titers were evaluated for binding in HEK293 cells stably expressing hRXFP2, rRXFP2, and parentals using flow cytometry.Three days after the final boost with either 25 µg human LDLa-linker-ratFc and 25 µg rat LDLa-linker-ratFc or 1e 6 irradiated rRXFP2 stable cells (Rad Source RS-2000), mice were euthanized and spleens and lymph nodes were harvested.All mice maintained health throughout the study.Euthanasia was performed following the American Veterinary Medical Association guidelines.Mice were euthanized via carbon dioxide inhalation (CO 2 ), using 100% CO 2 gas at an air displacement rate of 30-70% per minute, followed by cervical dislocation.

Figure 1 .
Figure 1.RXFP2 is associated with resistant hypertension in men.(a) Manhattan plot of GWAS on resistant hypertension versus non-resistant hypertension in the 23andMe cohort.Locuszoom plots of the association upstream of RXFP2 in both men and women (b) and in women and men separately (c).The lead SNP (rs2146377) is highlighted.(d) RXFP2 allelic status is associated with RXFP2 gene expression changes in the adrenal gland (GTEx v8, www.gtexp ortal.org/ home/ snp/ rs214 6377, p = 1.4e-14).(e) Endogenous RXFP2 mRNA expression relative to beta-actin in adrenal gland of hypertensive men (n = 6) is increased compared to normotensive men (n = 6, p = 0.041, Mann-Whitney U test).

Figure 3 .
Figure 3. Characterization of RXFP2 mAbs in screening and profiling assays.(a) ELISA and cell binding data showing the binding distribution of all antibodies generated against RXFP2.The majority of antibodies generated bind to the LDLa-linker domain.(b) Biacore S200 ka vs kd binding data against rat LDLa-linker protein.(c) Biacore S200 ka vs kd binding data against human LDLa-linker protein.Three antibodies within the anti-RXFP2 panel show cross reactivity to the human LDLa-linker domain.KD values against human LDLalinker protein: 2.75 nM (4B1), 11.7 nM (4F6), and 2.91 nM (4G6).(d, e) Representative dose-response curves of RXFP2 mAb treatment in INSL3 (EC 80 )-stimulated cAMP assay in HEK cells stably expressing rat RXFP2 (d, n = 2) or human RXFP2 (e, n = 2).The INSL3 EC 80 of 13.96 nM and 2.92 nM were used, respectively.(f) Threepoint RXFP2 mAb selectivity screen for activity against relaxin-stimulated cAMP activity in HEK cells stably expressing rat RXFP1 (n = 2).(g) Three-point RXFP2 mAb agonism screen for RXFP2 mAb activity in cAMP assay in HEK cell stably expressing rat RXFP2 (n = 2).

Figure 6 .
Figure 6.Pharmacokinetic study of RXFP2 mAbs in rat.(a) Six RXFP2 mAbs (1G1, 2D4, 4B1, 4G6, 1F2, 3F3)were discretely dosed to male Wistar Han rats (1 mg/kg, s.c., n = 3/mAb) once and the plasma concentration of RXFP2 mAbs was determined over 28 days.(b) Rat pharmacokinetic parameters calculated by noncompartmental analysis (NCA) analysis.Values are reported in mean ± standard deviation.ND: not determined due to insufficient data points or AUC extrapolated% > 20.C max is the maximum concentration, T max is the time to the maximum concentration, T 1/2 is the terminal half-life, Cl/F is the apparent clearance, AUC last is the area under the concentration-time profile from time zero to the time of last quantifiable concentration.

Table 1 .
Discovery of small molecule RXFP2 antagonists.Summary of RXFP2 small molecule blockers in screening and profiling assays.Table shows means of IC 50 and max response for INSL3-stimulated cAMP activity in HEK cells with Bacmam expression of human RXFP2 (hRXFP2), rat RXFP2 (rRXFP2), or human RXFP1.N is the number of tests in the assay; LE is ligand efficiency; LLE is lipophilic ligand efficiency; MWt is molecular weight, CAD SOL is solubility; NT is not tested; NA is no result since calculation is not accurate if compound activity is > 100 uM.

Table 2 .
Discovery of RXFP2 monoclonal antibodies.Summary of RXFP2 antibody characteristics in LDLlinker binding assay, INSL3-simulated cAMP activity assays in HEK cells stably expressing either human RXFP2 or rat RXFP2, and INSL3-stimulated CYP11B1 mRNA and CYP11B2 mRNA expression in H295R stably expressing rat RXFP2.NB = No binding occurred.ND = Not determined due to insufficient data points.N = 2 per assay.